Fluorescent dyes have many uses and are known to be particularly suitable for biological applications in which the high detectability of fluorescence is required. Fluorescent dyes are used to impart both visible color and fluorescence to other materials.
Fluorescence useful for such applications is generally initiated by absorption of light from an external, relatively concentrated light source. The sensitivity of these applications is improved by having dyes that have high absorbance of the exciting light and high fluorescence quantum yield. The applications are furthermore improved by having dyes that resist photobleaching by the exciting light and that have spectral wavelengths in a range that avoids the background from contaminants that may be present in the samples. For many biological applications it is useful to have dyes whose fluorescence is not quenched by water, since most biological measurements are made in aqueous solution.
Certain lasers are particularly useful as a concentrated light source for the excitation of fluorescence. The argon laser has been the most common light source for excitation of fluorescence, with principal output at 488 nm and 514 nm. Now other lasers are increasingly used, such as helium-neon lasers that can be selected to have maximum output at either 543 nm, 594 nm, or 633 nm; the krypton laser which has significant output at 568 nm and 647 nm; and light emitting diodes which are available at this time with output commonly above 660 nm; resulting in increased demand for longer wavelength fluorescent dyes.
A number of dyes that have previously been found to be fluorescent do not have significant absorbance at desired longer excitation wavelengths. Many also have other characteristics which interfere with or limit their usefulness. For example, many known fluorescent dyes are significantly quenched in aqueous solution or are unstable during the illumination.
Dyes derived from dipyrrometheneboron difluoride have many desirable characteristics. Simple alkyl derivatives of the fluorophore 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene have been described by Treibs & Kreuzer, Difluorboryl-komplexe von di- und tripyrrylmethenen, LIEBIGS ANNALEN CHEM. 718, 203 (1968) and by Worries, Kopek, Lodder, & Lugtenburg, A novel water-soluble fluorescent probe: Synthesis, luminescence and biological properties of the sodium salt of the 4-sulfonato-3,3',5,5'-tetramethyl-2,2'-pyrromethen-1,1'-BF.sub.2 complex, RECL. TRAV. CHIM. PAYS-BAS 104, 288 (1985) as being highly fluorescent with spectral properties that are similar to fluorescein, with maximum absorbance at about 490 to 510 nm and maximum emission at about 500 to 530 nm.
U.S. Pat. No. 4,774,339 to Haugland et al. (1988) ('339 patent) describes 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (dipyrrometheneboron difluoride) dyes including hydrogen, halogen, alkyl, cycloalkyl, aryl, arylalkyl, acyl and sulfo-substituted derivatives that contain reactive groups suitable for conjugation to biomolecules, that have good photostability, and which have fluoresceinlike spectra.
As described in the '339 patent, and by Pavlopoulos, et al., Laser action from a tetramethylpyrromethene-BF.sub.2 complex, APP. OPTICS 27, 4998 (1988), the emission of the alkyl derivatives of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene fluorescent dyes clearly overlaps that of fluorescein. The overlap allows the alkyl derivatives of dipyrrometheneboron difluoride to be used with the same optical equipment as used with fluorescein-based dyes without modification of the excitation sources or optical filters. As a result of having the same spectral characteristics, however, the fluorescence of the known class of alkyl-substituted 4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes is not readily suitable for detection in combination with fluorescein or for use in applications where excitation by longer wavelength sources such as the helium-neon or krypton lasers or light emitting diodes is required.
U.S. Pat. No. 4,916,711 to Boyer, et al. (1990) ('711 patent) discloses a method of using derivatives of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dyes, in particular symmetrical alkyl and sulfonated alkyl derivatives, as laser dyes. The '711 patent also discloses a multitude of possible alternatives for substituents of the basic tricyclic structure which can be used for the patented method. The '711 patent, however, is neither enabling nor prior art for the invention of ethenyl-substituted dipyrrometheneboron difluoride dyes. The '711 patent neither recognizes nor recites the effect or advantage of the alkenyl substitution in enhancing the long wavelength fluorescence properties of the dyes.
The novel dyes described in this invention contain one or more ethenyl groups directly coupled to the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene fluorophore through a single covalent bond ("ethenyl BDI dyes"). The ethenyl BDI dyes have significant absorbance and fluorescence at desired longer wavelengths, high fluorescence in aqueous solution and good photostability and are thus particularly useful as fluorescent dyes. Furthermore, the subject fluorescent dyes are desirable for use in combination with other fluorescent dyes such as fluorescein or alkyl-substituted 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dyes in that their fluorescence can be both selectively excited and detected because of their spectral shift to longer wavelengths, particularly an absorption maximum at greater than 525 nm and emission maximum at greater than 550 nm .